The pool of fatty acids covalently bound to platelet proteins by thioesterlinkages can be altered by exogenously supplied fatty acids

The goals of this investigation were, first, to develop a chemical strategy to identify and quantitate the mass of fatty acid which is covalently boun d to proteins by thioester linkage in unactivated platelets, and, second, t o determine whether exogeneously added fatty acids can alter the fatty acid composition of thioester bound fatty acids. Studies with radiolabeled fatt y acids cannot identify and quantitate the actual fatty acids bound to prot eins because they permit;analysis of only the radiolabeled fatty acids adde d and their metabolites. Therefore, in the absence of metabolic labeling by radiolabeled fatty acids, we isolated the thioester-linked fatty acids fro m platelet proteins using hydroxylamine at neutral pH to form fatty acid hy droxamates. The hydroxamates were subsequently converted to fatty acid meth yl esters by acid methanolysis for quantitation by gas chromatography-mass spectrometry. Using platelet specimens from 14 subjects, 74% of the fatty a cid recovered from the unactivated platelet proteins as thioester linked wa s palmitate. Importantly, however, 22% was stearic acid, and oleate was 4% of the total thioester bound fatty acid. There was minimal variability (2.6 -fold at maximum) between the subjects in the amount of the thioester-linke d palmitate and thioester-linked stearate. However, there was substantial v ariability (>100-fold at maximum) between subjects in the amount of thioest er-linked oleate. We also demonstrated that incubation of platelets with ex ogenous fatty acids can alter the profile of fatty acids bound to platelet proteins by thioester linkages. Incubation of platelets with 100 mu M palmi tate for 3 h increased the amount of thioester-linked palmitate by up to 26 %, and incubation of platelets with 100 CIM stearate increased the amount o f thioester;linked stearate up to 30%. In support:of the observation that r adiolabeled fatty acids other than palmitate were shown to be capable of bi nding to platelet proteins by thioester linkage, our results indicate that the fatty acids actually bound to unactivated platelet proteins include a s ignificant amount of stearate, and variable amounts of oleate, as well as p almitate. In addition, the data show that palmitate and stearate can be inc reased, as a percentage of total protein-bound fatty acid, by incubation wi th exogenous palmitate and stearate, respectively.